The quest for therapies able to induce tolerance in human transplantation remains unfulfilled. New strategies targeting T cell signaling molecules may induce transplant tolerance by qualitatively altering T cell activation. CD45 is a family of transmembrane PTPases critically involved in regulating T cell receptor-mediated signals. Multiple CD45 isoforms are differentially distributed on subsets of T cells having distinct functions. Anti-CD45 can induce tolerance in murine models of islet and renal transplantation. Preliminary data demonstrate that a short course of anti-CD45 as sole therapy, can induce long-term survival of renal allografts in non-human primates. Thus, CD45 is a potent immunotherapeutic target that clearly has potential in human islet transplantation. However, the mechanism(s) by which anti-CD45 acts are poorly understood. CD45 regulates key signals involved in the generation of anergy. Furthermore, treatment of mice with toleragenic anti-CD45 mAbs induces a shift in CD45 isoform expression towards the lower (CD45RBLO) Mr forms. T cells expressing these isoforms preferentially secrete Th2-type cytokines and downregulate autoimmunity in several animal models. Important lead data now indicate that the ant.i-CD45-mediated shift in CD45 isoforms is associated with an upregulation of CTLA-4, a potent inhibitory molecule essential for induction of peripheral tolerance. This utilizes an apparently novel signaling pathway that does not involve overt T cell activation. Moreover, unlike other agents that act through signal one, anti-CD45 synergizes with costimulatory blockade allowing prolongation of graft survival in stringent murine allograft models including skin transplantation. The studies proposed will extend our understanding of the mechanisms by which anti-CD45RB acts, and determine how costimulatory blockade synergizes with anti-CD45. In Aim 1, in vivo studies in murine islet transplantation will determine whether CTLA-4 plays a specific role in anti-CD45RB-mediated engraftment. In Aim 2, we will use both in vivo and in vitro studies to delineate the biochemical effects of CD45 ligation and determine the signaling pathways between CD45 and CTLA-4 expression. In Aim 3, we will determine the roles of anergy and apoptosis in anti-CD45RB-mediated engraftment. In Aim 4, we will determine whether combined therapy with anti-CD45, anti-CD4OL and DST can generate tolerance to primary skin grafts and based on the findings above, we will determine the basis for synergy by these agents. These studies will elucidate the mechanism of action of anti-CD45, supporting the initiation of clinical trials in human islet transplantation, and will provide novel insight into tolerance induction thaL may allow the development of new therapeutic strategies in the future.